The Human α-Type Proteasomal Subunit HsC8 Forms a Double Ringlike Structure, but Does Not Assemble into Proteasome-like Particles with the β-Type Subunits HsDelta or HsBPROS26

Abstract

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different ␣-and ␤-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human ␣-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant ␣-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form ␣-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human ␤-type subunits, HsBPROS26 and HsDelta. Both these ␤-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the ␣-and ␤-subunit of T. acidophilum, coexpression of the human ␤-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

The α4 and α7 subunits and assembly of the 20S proteasome

“…However, the expression of human a1 or a6 subunits results in the formation of dimers only [15]. It has also been shown that the expression of Trypanosoma brucei a5 subunit results in the formation of a complex of four heptameric rings [14].…”

“…Similarly, two other a-subunits HsPROs 27 (a1) and HsPROs 30 (a6) can also form protein dimers [15]. In addition, human a7 protein can induce hetero-oligomeric ring formation when coexpressed with a1 and a6 subunits, which are adjacent to a7 in each a-subunit ring of 20S proteasome [15].…”

“…Moreover, when expressed in Escherichia coli, some recombinant eukaryotic a-subunits including the human a7 (HsC8) and Trypanosoma brucei a5 (f-subunit) self assemble into double and even four stacked protein rings [13,14]. Similarly, two other a-subunits HsPROs 27 (a1) and HsPROs 30 (a6) can also form protein dimers [15]. In addition, human a7 protein can induce hetero-oligomeric ring formation when coexpressed with a1 and a6 subunits, which are adjacent to a7 in each a-subunit ring of 20S proteasome [15].…”

The proteasome: a protein‐destroying machine

“…However, the co-expression of both subunits results in fully assembled proteolytically active proteasomes. Analogously, certain human ␣-type subunits form a double ring-like structure, but the ␤-type subunits are not assembled into a large particle (Gerards et al 1997). The latter observation, however, is puzzling when considering the assembly of native proteasomes, because eukaryotic proteasomes are hetero-heptameric complexes.…”

Proteasomes in the archaea: from structure to function

“…Some eucaryal alpha subunits including the human alpha7 (HsC8) and Trypanosoma brucei alpha5 proteins self-assemble into single, double, and even four-stacked protein rings when produced in recombinant E. coli (168,169). The human alpha7 protein also induces heteroligomeric ring formation when synthesized with alpha1 (PROS27) and alpha6 (PROS30) which are adjacent to alpha7 in the final ring of the 20S proteasome (170).…”